American Academy of Microbiology

Education Resources

Laboratory Protocols

Laboratory Protocols are information briefs about standard laboratory tests and include the procedural steps as well as the purpose, theory, history, safety considerations, tips and comments, and references. The protocols are geared for microbiology educators at all levels and assume a basic understanding of general microbiology. All protocols are free, original, open access, and peer-reviewed by the microbiology educator community. Protocols were published between 2005 and 2015.

Agglutination is the reaction between surface antigens of bacteria and antigen-specific antibodies. The agglutination reaction is a useful tool both in identifying bacterial isolates and diagnosing infection through the detection of bacterial-specific antibodies in samples.

The membrane filtration technique is used to examine water samples from different sources. The membrane is incubated on an agar plate. Bacterial (and other) cells trapped on the membrane will grow into colonies that can be counted, and a bacterial density of the water samples can be calculated.

Carbohydrate fermentation tests aid in determining the ability of microorganisms to ferment a specific carbohydrate. Fermentation patterns can be used to differentiate among bacterial groups or species. Fermentation reactions are detected by the color change of a pH indicator as acid products are formed. A color change only occurs when enough acid products have been produced by fermentation of the carbohydrate to lower the pH to 6.8 or less. Another by-product of fermentation is gas, which may be hydrogen or carbon dioxide. If a Durham tube is added to the fermentation broth, the presence of a gas bubble at the top of the tube is another indication that fermentation of the carbohydrate has taken place. While fermentation tests can be performed on microorganisms other than bacteria, this protocol only addresses fermentation of carbohydrates by bacteria.

The catalase test is used to detect the presence of the enzyme catalase in bacteria. Catalase serves to neutralize the bactericidal effects of hydrogen peroxide. Its concentration in bacteria has been correlated with pathogenicity. This enzymatic test is essential in the scheme of identification for gram-positive organisms and certain gram-negative organisms. It is a primary test used in the differentiation of staphylococci and streptococci.

This protocol describes the history and procedures of the coagulase test. The coagulase test is used to differentiate species of Staphylococcus, especially the coagulase-positive Staphylococcus aureus from coagulase-negative staphylococcal species. Both common versions of the test, the slide method and the test tube method, are described, and the mechanisms of the reactions are discussed.

The decarboxylase media, used to identify bacteria's ability to decarboxylate amino acids, were first introduced by Moeller for detecting lysine and ornithine decarboxylase and arginine dihydrolase among bacteria belonging to Enterobacteriaceae. The Moeller's basal media contained peptone, beef extract, bromocresol purple, cresol red, pyridoxal, and glucose. To test decarboxylase activity, 1% of the appropriate amino acid was added. The medium was poured in narrow tubes as a column of about 2 cm in height and autoclaved, after which a layer of about 5 mm sterile paraffin oil was poured in each tube. The decarboxylase activity was measured onthe basis of a pH rise of the amino acid reagent that was made visible by an indicator. After incubation the color changes in the tubes were followed for up to 10 days.

The gelatin hydrolysis test is used to detect the ability of microorganisms to produce the enzyme gelatinase. This test is helpful in identifying and differentiating species of Bacillus, Clostridium, Proteus, Pseudomonas, and Serratia. In this protocol, the history, theory, procedure, and interpretation of results will be discussed in detail.

Hektoen enteric agar is a selective and differential media for the recovery of enteric gram-negative rods from mixed microbiota. The growth of gram-positive organisms and nonpathogenic enteric coliforms is inhibited through the use of bile salts and dyes, allowing intestinal pathogens, such as Salmonella and Shigella, to be more easily recovered. The media can also differentiate between organisms that produce H2S and those that do not due to the presence of an iron-containing compound. The use and interpretation of growth on this media is discussed in this protocol.

Motility test medium is used to determine the motility of bacteria. The motility test is used in the identification of bacteria since motility has long been recognized as an important taxonomic tool and biological characteristic of bacteria.

The nitrate and nitrite reduction test determines the ability of bacteria to produce the enzyme nitrate reductase and reduce nitrate and/or nitrite. The nitrate and nitrite test is useful for the identification of bacteria and separating members of the family Enterobacteriaceae from other gram-negative bacilli.

The oxidase test is used to detect the presence of the enzyme cytochrome oxidase in microorganisms. While used as a taxonomic tool for many microorganisms, the test was established initially to differentiate Neisseria spp. (oxidase positive) from Acinetobacter (oxidase negative) and Pseudomonas spp. (oxidase positive) from the Enterobacteriaceae (oxidase negative).

Phenylethyl alcohol agar (PEA) is a selective medium that permits the growth of gram-positive cocci while inhibiting most gram-negative organisms. PEA agar is used for the isolation of gram-positive Staphylococcus species and Streptococcus species from clinical specimens or specimens that contain mixtures of bacterial flora.

Starch agar is a differential medium that tests the ability of an organism to produce the extracellular enzymes a-amylase and oligo-1,6-glucosidase that hydrolyze starch. In this protocol, the history, procedure, and interpretation of results of this useful agar medium are discussed in detail.

Production of the enzyme urease is characteristic of the Proteeae and a few other members of the Enterobacteriaceae among others. Urease is a constitutively produced enzyme that hydrolyzes urea with the resultant release of ammonia and carbon dioxide. The use of urea agar and urea broth for the detection of urease activity is described in this protocol.

Escherichia coli broth and Escherichia coli agar media with 4-methylumbelliferyl-ß-D-glucuronide are used to confirm the presence of Escherichia coli in water samples. In this protocol, the history, procedure, and interpretation of results of this useful technique are discussed in detail.